Rifampin (RIF), an antibiotic synthesized from rifamycin B, is a key component of drug therapy against Mycobacterium tuberculosis. Rifampin has a unique site of action on the beta subunit of prokaryotic RNA polymerase. In Escherichia coli, missense mutations and short deletions in the central region of the RNA polymerase subunit gene (rpoB) result in strains resistant to rifampin (Lisityn et al., 1984, Mol. Gen. Genet. 196 :173–174). Similarly, in M. tuberculosis a wide variety of mutations in the rpoB gene have been identified that confer rifampin resistance (Telenti et al., 1993, Lancet 341: 647–650). More than 90% of rifampin-resistant M. tuberculosis isolates are also resistant to isoniazid, and, therefore, rifampin resistance is a valuable surrogate marker for multiple drug resistance. Thus, there is a need for tests that can detect rapidly the genetic basis for rifampin resistance for diagnosis that leads to appropriate treatment of infected individuals.
Early detection of drug resistance in clinical M. tuberculosis isolates is crucial for appropriate treatment and to prevent the spread of resistant strains. Conventional methods of detecting drug-resistance by growth of M. tuberculosis on solid media, and more recent methods that rely on growth in liquid media have provided susceptibility results in 3 days to over 4 weeks (Rusch-Gerdes et al., 1999, J. Clin. Microbiol. 37: 45–48).
Genetic techniques that rely on the polymerase chain reaction (PCR) have been devised to detect rifampin resistance. Such techniques include direct sequencing of PCR products, single strand conformation polymorphism analysis, heteroduplexing and dideoxy fingerprinting (Telenti et al., 1993, Lancet 342: 841–844; Williams et al., 1994, Antimicrobial Agents Chemotherapy 38: 2380–2386; De Beenhouwer, 1995, Tubercle and lung disease 76: 425–430). Other assays and reagents for detecting resistance to rifampin in M. tuberculosis isolates or identifying Mycobacteria species using rpoB gene have been previously disclosed, for example, in U.S. Pat. No. 5,643,723 (Persing et al.), U.S. Pat. No. 5,851,763 (Heym et al.), U.S. Pat. No. 6,228,575 (Gingeras et al.), and 6,242,584 (Kook et al.).
The present invention provides compositions and simple diagnostic methods to detect rifampin resistance in M. tuberculosis that may be present in a clinical sample.